Cell Fusion
The ability to fuse together artificially two types of cells to generate a third cell, which displays hybrid characteristics of the two unfused cells, plays a central role in biotechnology. The development of monoclonal antibodies by Koehler and Milstein [see e.g. Kohler, G. & Milstein, C. 1975, Nature, 256, 495–497] for example, relies on the formation of “hybridomas” created by fusing antibody-producing cells with cancerous cells. The cloning of the sheep Dolly by Wilmut and coworkers [Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., Campbell, K. H. S. 1997, Viable offspring derived from fetal and adult mammalian cells, Nature, 385, 810–813] provides another example where the fusion between two types of cells (an adult cell from the mammary gland with an egg cell) played an essential role.
There are a number of methods for carrying out cell-cell fusion in vitro, including the use of chemicals such as polyethylene glycol (PEG), the use of focused laser beams (laser-induced fusion), and the application of pulsed electric fields (electrofusion). Of these methods, electrofusion has developed into an extremely efficient method for the fusion of mammalian cells, mainly because of its mild conditions, which result in a high number of viable fusion products [see e.g. White, K. L. 1995, Methods in Molecular Biology, 48, 283–293]. The application of an electrical field over phospholipid bilayer membranes induces pore formation when the applied potential reaches or exceeds the membrane breakdown potential. Consequently, electro-permeabilization techniques has been used in a wide variety of biological experiments, like electrofusion for the creation of hybridomas and new cell lines [see e.g. Zimmermann, U., et al., 1985, Adv. Biotechnol. Proc. 4, 79–150; Neil, G. A. et al., 1993. Electrofusion, Methods in Enzymology, 220, 174–196; Glassy, M. 1988, Nature, 333, 579–580], in vitro fertilization [see e.g. Ogura, A. et al., 1995, Reprod. Fertil. Dev., 7, 155–159], cloning experiments [see e.g. Van Stekelenburg-Hamers, A. E. P., et al., 1993, Mol. Reprod. Dev., 36, 307–312], [see e.g. Li, H., et al., 1997, J. Neurosci. Methods, 75, 29–32; Lundqvist, J. A., et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 10356–10360], and electroinsertion for the addition of membrane-associated macromolecules, including proteins [see e.g. Mouneimne, Y., et al., 1989, Electroinsertion of xeno-glycophorin into the red blood cell membrane, Biochem. Biophys. Res. Com. 159, 34–40]. Applications of in vivo electrofusion include the incorporation of gonococcal attachment receptors from human HL60 cells to rabbit corneal epithelial tissue as a viable model of human-specific pathogens [see e.g. Heller, R., et al., 1990, Transfer of human membrane surface components by incorporating human cells into intact animal tissue by cell-tissue electrofusion in vivo, Biochim. Biophys. Acta, 1024, 185–188].
Electric-field-induced fusion is widely employed in biomedical research for a population of cells in suspension. Cells are first brought into contact by dielectrophoresis through the application of a low-amplitude, high-frequency AC field and subsequently a fraction of the cells are fused by a strong and short DC pulse. Although bulk electrofusion of large quantities of cells is useful for creating and selecting new cell lines, it cannot be applied to fuse single cells with high precision. The main drawback of carrying out bulk electrofusion (or PEG-induced fusion) inside a fusion chamber is the inability to manipulate and fuse individual cell pairs selectively based on the biological characteristics of the cells; dielectrophoresis brings together cells and align them into pearl chains inside the fusion chamber based on cell polarization. Practical constraints also limit cell fusion to two cell types at a time inside the fusion chamber. Based on statistics, this random fusion between two cell types of equal concentrations will result in 50% heterokaryon formation (fusion of different cell types) and 50% homokaryon formation (fusion of same cell types). In practice, however, cells of different types often align into pearl chains in segregation because of their differences in size and polarization; provided there are “enough” cells within the fusion chamber, there is usually enough intermixing to ensure heterocytotic fusion. It is, therefore, difficult to carry out cell fusion selectively and controllably among many different cell types using this traditional format. To overcome partially some of the drawbacks of this format, several approaches were developed to achieve cell pair selection prior to fusion, including the use of ligand-receptor recognition, flow cytometry, and laser-based single-cell manipulation. In this invention, we describe a new method based on the use of microfluidic channels for the combinatorial fusion of cells and liposomes.
Electroporation
It has for a long time been recognised that cell membranes can be permeabilised by pulsed electric fields (see e.g. Zimmermann, U. Biochim. Biophys Acta, 694, 227–277 (1982) This technique is called electroporation. The membrane voltage, Vm, at different loci on phospholipid bilayer spheres during exposure in a homogenous electric field of duration t, can be calculated from:Vm=1.5rcE cos α[1−exp(−τ/t)]  (1)where E is the electric field strength, rc is the cell radius, α, the angle in relation to the direction of the electric field, and t the capacitive-resistive time constant. Pore-formation will result at spherical coordinates exposed to a maximal potential shift, which is at the poles facing the electrodes (cos α=1 for α=0; cos α=−1 for α=π). Generally, electric field strengths on the order of from 1 to 1.5 kV/cm for durations of a few μs to a few ms are sufficient to cause transient permeabilisation in 10-μm-outer diameter spherical cells.
Traditionally, electroporation is made in a batch mode allowing for administration of polar solutes into several millions of cells simultaneously. The electrodes producing such fields can be several square centimetres and the distance between the electrodes several centimetres, thus requiring high-voltage power sources to obtain the needed electrical field strength to cause electrically induced permeabilisation of biological membranes.
Instrumentation that can be used for electroporation of a small number of cells in suspension (K. Kinosita, Jr., & T. Y. Tsong, T. Biochim. Biophys. Acta, 554, 479–497(1979); D. C Chang, J. Biophys., 56, 641–652 (1989; P. E. Marszalek, B. Farrel, P. Verdugo, & J. M. Fernandez, Biophys. J., 73, 1160–1168 (1997)) and for a small number of adherent cells grown on a substratum (Q. A. Zheng, & D. C. Chang, Biochim. Biophys. Acta, 1088, 104–110 (1991); M. N. Teruel, & T. Meyer Biophys. J., 73, 1785–1796 (1997)) have also been described. The design of the electroporation device constructed by Marszalek et al. is based on 6 mm long 80 μm diameter platinum wires that are glued in a parallel arrangement at a fixed distance of 100 μm to a single glass micropipette. The design by Kinosita and Tsong uses fixed brass electrodes spaced with a gap distance of 2 mm, the microporator design of Teruel and Meyer relies on two platinum electrodes that are spaced with a gap distance of about 5 mm, and the electroporation chamber design by Chang uses approximately 1 mm-long platinum wires spaced at a distance of 0.4 mm. It is obvious, that these electroporation devices create electric fields that are several orders of magnitude larger than the size of a single cell which typically is 10 μm in diameter, and thus can not be used for exclusive electroporation of a single cell. They are also not optimal for use in a micofluidic device.
Microfluidics
Microfluidic systems provide an attractive and versatile platform for the manipulation, isolation, and transport of selected cells prior to either electric-field induced manipulation of cells or by manipulation of cells by other means, such as chemical-induced, or laser-induced fusion of cells. Microfabricated networks of micron-sized channels have already proven useful for applications in chemical separations (e.g. capillary electrophroesis), biochemical assays, DNA analyses, medical diagnostics, drug delivery, and cell manipulation and sorting. The ease with which arrays of microelectrodes can be patterned and integrated with networks of microchannels makes microfluidic systems an especially attractive platform for applications in electrofusion in which fusion among a multitude of different cell types is desired.
It is time consuming and impractical to use bulk fusion and electroporation chambers for applications involving a large number of cell types and fusion or electroporation events. With chip-based microfluidic systems, however, a large number of cells can be transported, combined, separated, and sorted; cell fusions and electroporations can be achieved on-chip either in parallel or in rapid sequence. In addition, the amount of cells required for each cell type may be dramatically reduced owing to the reduced volumes in the microchannels (in comparison with fusion or elcetroporation chambers) provided the cell fusion or electroporation and survival yields are acceptable.
Liposomes
Liposomes are synthetic lipid-bilayer containers that can be used to mimic the surface properties of natural biological compartments. The sizes of these containers can also be varied from tens of nanometers to tens of micrometers in diameter (which correspond to 10−21 to 10−12 L) for mimicking the natural size distribution of organellar and cellular compartments. These versatilities together with the ease that protein functions can be reconstituted in liposomes make them attractive as reaction containers for approximating the in vivo conditions where biochemical reactions occur [see e.g. Chakrabarti, A. C.; Breaker, R. R.; Joyce, G. F.; Deamer, D. W. J. Mol. Evol. 1994, 39, 555–559; Oberholzer, T.; Albrizio, M.; Luisi, P. L. Chem. Biol. 1995, 2(10), 677–682; Oberholzer, T.; Wick, R.; Luisi, P. L.; Biebricher, C. K. Biochem. Biophys. Res. Commun. 1995, 207(1), 250–257; Steinberg-Yfrach, G.; Rigaud, J. L.; Durantini, E. N.; Moore, A. L.; Gust, D.; Moore, T. A. Nature, 1998, 392(6675), 479–482; Oberholzer, T.; Meyer, E.; Amato, I.; Lustig, A.; Monnard, P. A. Biochim. Biophys. Acta 1999, 1416, 57–68]. Reactions in these systems have been measured, typically, as an ensemble average from millions of liposomes and may therefore hide interesting information concerning reaction rates and mechanisms. Recently, methods have been developed where chemical reactions can be initiated in single liposomes using microinjection [see e.g. Bucher, P.; Fischer, A.; Luisi, P. L.; Oberholzer, T.; Walde, P. Langmuir 1998, 14(10), 2712–2721], electroinjection, electroporation [see e.g. Miyata, H.; Nishiyama, S.; Akashi, K.; Kinosita, K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96(5), 2048–2053; Chiu, D. T.; Wilson, C. F.; Ryttsén, F.; Strömberg, A.; Farre, C.; Karlsson, A.; Nordholm, S.; Gaggar, A.; Modi, B. P.; Moscho, A.; Garza-López, R. A.; Orwar, O.; Zare, R. N. Science 1999, 283, 1892–1895], and electrofusion [see e.g. Chiu, D. T.; Wilson, C. F.; Ryttsén, F.; Strömberg, A.; Farre, C.; Karlsson, A.; Nordholm, S.; Gaggar, A.; Modi, B. P.; Moscho, A.; Garza-López, R. A.; Orwar, O.; Zare, R. N. Science 1999, 283, 1892–1895]. In combination with single-molecule detection techniques [see e.g. Nie, S., Zare, R. N., Annu. Rev. Biophys. Biomol. Struct., 1997, 26, 567], these single-liposome manipulation methodologies provide a powerful set of tools for studying chemical reactions inside liposomes at a level of detail that was previously unattainable [see e.g. Chiu, D. T.; Wilson, C. F.; Ryttsén, F.; Strömberg, A.; Farre, C.; Karlsson, A.; Nordholm, S.; Gaggar, A.; Modi, B. P.; Moscho, A.; Garza-López, R. A.; Orwar, O.; Zare, R. N. Science 1999, 283, 1892–1895; Chiu, D. T.; Wilson, C. F.; Karlsson, A.; Danielsson, A.; Lundqvist, A.; Strömberg, A.; Ryttsén, F.; Davidson, M.; Nordholm, S.; Orwar, O.; Zare, R. N. J. Chem. Phys. 1999, 247, 133–139].
In order to understand chemical behaviors relating both to intrinsic properties of the reaction system as well as to properties of the reaction surroundings, and to be able to optimize conditions for a given liposome-confined reaction, design and knowledge of the liposome reactor is of central importance. Container size, topography, surface charge, wetability as well as the phospholipid and membrane protein composition of a liposome reactor are some important properties that needs to be controlled. A seemingly simple issue such as variations in compartment size might have profound effects on both reactivity and mechanisms for a given enzyme-catalyzed reaction [see e.g. Chiu, D. T.; Wilson, C. F.; Karlsson, A.; Danielsson, A.; Lundqvist, A.; Strömberg, A.; Ryttsén, F.; Davidson, M.; Nordholm, S.; Orwar, O.; Zare, R. N. J. Chem. Phys. 1999, 247, 133–139; Mikhailov, A., Hess, B., J. Phys. Chem. 1996, 100, 19059–19065].